The structural disorder of intrinsically unstructured proteins is the outcome of a complex ensemble of conformers driven by a rugged energy landscape. Traditional bulk biochemical experiments mask this complexity in their inherent ensemble averaging. Human α-Synuclein is an intrinsically unstructured protein whose aggregation is involved in Parkinson’s disease. By mechanically stretching single molecules of α-Synuclein and recording their mechanical properties by the AFM-based Single Molecule Force Spectroscopy methodology we characterized the folding and the conformational diversity of this protein, a natively unstructured protein involved in Parkinson disease,. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the α-Synuclein sample, including disordered and “beta-like” structures. We found that this class of “beta-like” structures is directly related to α-Synuclein aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of £Syn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of α-Synuclein with a “beta-like” structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents. Also in the case of the structured proteins the identification of the active form might require a single-molecule approach, as demonstrated by us for angiostatin, a multimodular protein with antitumor activity. The AFM-based single-molecule-force spectroscopy provided evidence that in-vivo, on the surface of a tumor cell, this protein is not present with its native structure but with a partially reduced and unfolded structure whose access is controlled by a hierarchical coupling of redox and mechanical switches. This kind of result addresses very general questions like: “is the active form of a structured protein only ever the native one?” and sets boundaries for the paradigm “one sequence, one structure, one function”. (ChemBioChem 2006,7, 1774-1782)

Catching the active form of a protein by single molecule methodologies

SAMORI', BRUNO
2007

Abstract

The structural disorder of intrinsically unstructured proteins is the outcome of a complex ensemble of conformers driven by a rugged energy landscape. Traditional bulk biochemical experiments mask this complexity in their inherent ensemble averaging. Human α-Synuclein is an intrinsically unstructured protein whose aggregation is involved in Parkinson’s disease. By mechanically stretching single molecules of α-Synuclein and recording their mechanical properties by the AFM-based Single Molecule Force Spectroscopy methodology we characterized the folding and the conformational diversity of this protein, a natively unstructured protein involved in Parkinson disease,. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the α-Synuclein sample, including disordered and “beta-like” structures. We found that this class of “beta-like” structures is directly related to α-Synuclein aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of £Syn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of α-Synuclein with a “beta-like” structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents. Also in the case of the structured proteins the identification of the active form might require a single-molecule approach, as demonstrated by us for angiostatin, a multimodular protein with antitumor activity. The AFM-based single-molecule-force spectroscopy provided evidence that in-vivo, on the surface of a tumor cell, this protein is not present with its native structure but with a partially reduced and unfolded structure whose access is controlled by a hierarchical coupling of redox and mechanical switches. This kind of result addresses very general questions like: “is the active form of a structured protein only ever the native one?” and sets boundaries for the paradigm “one sequence, one structure, one function”. (ChemBioChem 2006,7, 1774-1782)
Single molecule Biophysics, (Aspen Center for Physics, February 4-9, 2007)
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Samorì B.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/84604
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